Fourier analysis of the nuclear and cytoplasmic shapes of living two-cell murine embryos

Living two-cell mouse embryos were flushed out from the oviduct 17, 24 and 36 hours after fertilization in order to obtain cells in the G1S, early G2 and late G2 phases of the second cell cycle. The nuclei of the living cells were stained with Hoechst 33342. The coordinates of the contour shapes of the entire cells (cellular contours) were registered by contour image processing with a TV camera coupled with a computer; the contours of the nuclei were computed by means of a digitizer coupled with the computer. Fourier analysis of the cellular and nuclear contours revealed systematic modifications in the folding of the cells and nuclei in the course of the murine second cell cycle. The progression of cells through the second cell cycle was correlated with an increasing diversification of cellular and nuclear shape, with the diversification being much more pronounced in the nuclear shapes.     

Positioning and stability of mitotic spindle orientation in the neuroepithelial cell

To verify non-random positioning and to define the stability of the mitotic spindle orientation in neuroepithelial cells of mouse foetuses, computer - assisted morphometric analysis at the light microscopy level was performed. It was confirmed that the mitotic spindle axis is positioned non-randomly in relation to the cell polarity axis and could be displaced only within a narrow range. This orientation was found to be attained at metaphase and it does not change until telophase is completed. However, in relation to the long axis of the neural tube the mitotic spindle axis was found to be positioned randomly. In the light of these findings centrosome movement and positioning are discussed.     

High Frequency of Germline TP53 Mutations in a Prospective Adult-Onset Sarcoma Cohort

Sarcomas are a key feature of Li-Fraumeni and related syndromes (LFS/LFL), associated with germline TP53 mutations. Current penetrance estimates for TP53 mutations are subject to significant ascertainment bias. The International Sarcoma Kindred Study is a clinic-based, prospective cohort of adult-onset sarcoma cases, without regard to family history. The entire cohort was screened for mutations in TP53 using high-resolution melting analysis and Sanger sequencing, and multiplex-ligation-dependent probe amplification and targeted massively parallel sequencing for copy number changes. Pathogenic TP53 mutations were detected in blood DNA of 20/559 sarcoma probands (3.6%); 17 were germline and 3 appeared to be somatically acquired. Of the germline carriers, one appeared to be mosaic, detectable in the tumor and blood, but not epithelial tissues. Germline mutation carriers were more likely to have multiple cancers (47% vs 15% for non-carriers, P = 3.0×10⁻³), and earlier cancer onset (33 vs 48 years, P = 1.19×10⁻³). The median survival of mutation carriers following first cancer diagnosis was not significantly different from non-carriers. Only 10/17 (59%) pedigrees met classical or Chompret criteria for LFS. In summary, germline TP53 mutations are not rare in adult patients with sarcoma, with implications for screening, surveillance, treatment and genetic counselling of carriers and family members.     

Molecular analysis of three novel G6PD variants: G6PD Pedoplis-Ckaro, G6PD Piotrkow and G6PD Krakow

We present three novel mutations in the G6PD gene and discuss the changes they cause in the 3-dimensional structure of the enzyme: 573C-->G substitution that predicts Phe to Leu at position 191 in the C-terminus of helix alphae, 851T-->C mutation which results in the substitution 284Val--> -->Ala in the beta+alpha domain close to the C-terminal part of helix alphaj, and 1175T-->C substitution that predicts Ile to Thr change at position 392.     

Chronic Inflammation and Angiogenic Signaling Axis Impairs Differentiation of Dental-Pulp Stem Cells

Dental-pulp tissue is often exposed to inflammatory injury. Sequested growth factors or angiogenic signaling proteins that are released following inflammatory injury play a pivotal role in the formation of reparative dentin. While limited or moderate angiogenesis may be helpful for dental pulp maintenance, the induction of significant level of angiogenesis is probably highly detrimental. Hitherto, several studies have addressed the effects of proinflammatory stimuli on the survival and differentiation of dental-pulp stem cells (DPSC), in vitro. However, the mechanisms communal to the inflammatory and angiogenic signaling involved in DPSC survival and differentiation remain unknown. Our studies observed that short-term exposure to TNF-α (6 and 12 hours [hrs]) induced apoptosis with an upregulation of VEGF expression and NF-κB signaling. However, long-term (chronic) exposure (14 days) to TNF-α resulted in an increased proliferation with a concomitant shortening of the telomere length. Interestingly, DPSC pretreated with Nemo binding domain (NBD) peptide (a cell permeable NF-κB inhibitor) significantly ameliorated TNF-α- and/or VEGF-induced proliferation and the shortening of telomere length. NBD peptide pretreatment significantly improved TNF-α-induced downregulation of proteins essential for differentiation, such as bone morphogenic proteins (BMP)-1 & 2, BMP receptor isoforms-1&2, trasnforming growth factor (TGF), osteoactivin and osteocalcin. Additionally, inhibition of NF-κB signaling markedly increased the mineralization potential, a process abrogated by chronic exposure to TNF-α. Thus, our studies demonstrated that chronic inflammation mediates telomere shortening via NF-κB signaling in human DPSC. Resultant chromosomal instability leads to an emergence of increased proliferation of DPSC, while negatively regulating the differentiation of DPSC, in vitro.     

Heterogeneity of cytoplasmic and nuclear shape of lymphocytes during progression of chronic lymphocytic leukemia

Peripheral blood from ten healthy subjects and from 44 patients at stages 0, I, II, III, IV of chronic lymphocytic leukemia (CLL), type B, was routinely smeared, fixed and stained by the May-Grunwald-Giemsa method. Fourier analysis of nuclear and cytoplasmic shape of smeared lymphocytes was carried out for the range 1-20 of harmonics (describing the pattern of contour folding in quantitative terms). In addition the roughness coefficients (describing the summarized measure of contour folding of an individual cell) were calculated and computer evaluated. Cytoplasmic contour shape of smeared lymphocytes in the 6-10 harmonic range discriminates well between lymphocytes of healthy subjects and those of each CLL stage. This discrimination was the result of richer folding of CLL lymphocytes. Nuclear contour shape of lymphocytes in the 6-10 harmonic range fails to discriminate between lymphocytes of healthy subjects and those of CLL, but it discriminates well between lymphocytes of various stages of CLL, with the exception of stages I/II and III/IV. When Fourier analysis was carried out on lymphocytes of combined stages I + II and III + IV, the shape differences were even more accentuated. We conclude that nuclear and cytoplasmic contour shape is a phenotypic feature of lymphocytes that is markedly modified in the course of CLL progression; this feature may be used as a new parameter in CLL.     

Investigating the potential role of genetic and epigenetic variation of DNA methyltransferase genes in hyperplastic polyposis syndrome

Background

Hyperplastic Polyposis Syndrome (HPS) is a condition associated with multiple serrated polyps, and an increased risk of colorectal cancer (CRC). At least half of CRCs arising in HPS show a CpG island methylator phenotype (CIMP), potentially linked to aberrant DNA methyltransferase (DNMT) activity. CIMP is associated with methylation of tumor suppressor genes including regulators of DNA mismatch repair (such as MLH1, MGMT), and negative regulators of Wnt signaling (such as WIF1). In this study, we investigated the potential for interaction of genetic and epigenetic variation in DNMT genes, in the aetiology of HPS.

Methods

We utilized high resolution melting (HRM) analysis to screen 45 cases with HPS for novel sequence variants in DNMT1, DNMT3A, DNMT3B, and DNMT3L. 21 polyps from 13 patients were screened for BRAF and KRAS mutations, with assessment of promoter methylation in the DNMT1, DNMT3A, DNMT3B, DNMT3L MLH1, MGMT, and WIF1 gene promoters.

Results

No pathologic germline mutations were observed in any DNA-methyltransferase gene. However, the T allele of rs62106244 (intron 10 of DNMT1 gene) was over-represented in cases with HPS (p<0.01) compared with population controls. The DNMT1, DNMT3A and DNMT3B promoters were unmethylated in all instances. Interestingly, the DNMT3L promoter showed low levels of methylation in polyps and normal colonic mucosa relative to matched disease free cells with methylation level negatively correlated to expression level in normal colonic tissue. DNMT3L promoter hypomethylation was more often found in polyps harbouring KRAS mutations (p = 0.0053). BRAF mutations were common (11 out of 21 polyps), whilst KRAS mutations were identified in 4 of 21 polyps.

Conclusions

Genetic or epigenetic alterations in DNMT genes do not appear to be associated with HPS, but further investigation of genetic variation at rs62106244 is justified given the high frequency of the minor allele in this case series.